In this study, mitochondrial transcript information from a recent EST project wasextended to obtain complete mitochondrial genome sequence for 5 tiger salamandercomplex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense,and A. dumerilii). For the first time, aspects of mitochondrial transcription in arepresentative amphibian are described, and then complete mitochondrial sequencedata are used to examine salamander phylogeny at both deep and shallow levels ofevolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481)and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrialgenome, respectively. Complete mitochondrial sequences for all species were rapidlyobtained by using long distance PCR and DNA sequencing. A number of genomestructural characteristics (base pair length, base composition, gene number, geneboundaries, codon usage) were highly similar among all species and to other distantlyrelated salamanders. Overall, mitochondrial transcription in Ambystoma approximatedthe pattern observed in other vertebrates. From the mapping of ESTs onto mtDNA it wasinferred that transcription occurs from both heavy and light strand promoters andcontinues around the entire length of the mtDNA, followed by post-transcriptionalprocessing. However, the observation of many short transcripts corresponding to rRNAgenes indicates that transcription may often terminate prematurely to bias transcriptionof rRNA genes; indeed an rRNA transcription termination signal sequence was observedimmediately following the 16S rRNA gene. Phylogenetic analyses of salamander familyrelationships consistently grouped Ambystomatidae in a clade containingCryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust resultsuggests a novel alternative hypothesis because previous studies have consistentlyidentified Ambystomatidae and Salamandridae as closely related taxa. Phylogeneticanalyses of tiger salamander complex species also produced robustly supported trees.The D-loop, used in previous molecular phylogenetic studies of the complex, was foundto contain a relatively low level of variation and we identified mitochondrial regions withhigher rates of molecular evolution that are more useful in resolving relationships amongspecies. Our results show the benefit of using complete mitochondrial genomeinformation in studies of recently and rapidly diverged taxa.